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United States Patent |
5,583,153
|
Brahn
|
December 10, 1996
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Use of taxol in the treatment of rheumatoid arthritis
Abstract
An improved method of suppression of a progressive, inflammatory,
autoimmune arthritis in a mammal involves the use of the drug Taxol. In
general, such a method comprises administering to a mammal having or
susceptible to arthritis Taxol in a pharmacologically acceptable carrier
capable of solubilizing Taxol in a dose sufficient to suppress at least
one symptom of arthritis selected from the group of inflammation,
swelling, abnormal neovascularization, bone erosion, and cartilage
erosion. The use of Taxol can be combined with the use of other
antiarthritic drugs, such as the angiogenesis inhibitor AGM-1470, to
produce a greater therapeutic effect than with either Taxol or the other
antiarthritic drug alone.
Inventors:
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Brahn; Ernest (Encino, CA)
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Assignee:
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Regents of the University of California (Oakland, CA)
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Appl. No.:
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319236 |
Filed:
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October 6, 1994 |
Current U.S. Class: |
514/449; 514/475 |
Intern'l Class: |
A61K 031/335 |
Field of Search: |
514/449,475
|
References Cited
Other References
Brahn et al; "Regression of Collagen-Induced Arthritis with Taxol, a
Microlubule Stabilizer", Arthritis and Rheumatism; vol. 37, 14.0.6, Jun.
1994, pp. 839-845.
S. J. Oliver et al., "Suppression of Collagen-Induced Arthritis Using an
Angiogenesis Inhibitor, AGM-1470, and a Microtubule Stabilizer, Taxol,"
Cell. Immunol. 157:291-299 (1994).
E. K. Rowinsky, "Taxol: A Novel Investigational Antimicrotubule Agent," J.
Nat. Cancer Inst. 82: 1247-1259 (1990).
C. M. Spencer & D. Faulds, "Paclitaxel: A Review of Its Pharmacodynamic and
Pharmacokinetic Properties and Therapeutic Potential in the Treatment of
Cancer," Drugs 48:794-847 (1994).
D. Bissett & S. B. Kaye, "Taxol and Taxotere--Current Status and Future
Prospects," Eur. J. Cancer 29A: 1228-1231 (1993).
J. G. Kuhn, "Pharmacology and Pharmacokinetics of Paclitaxel," Ann.
Pharmacother. 28: S15-S18 (1994).
D. S. Sonnichsen & M. V. Relling, "Clinical Pharmacokinetics of
Paclitaxel," Clin. Pharmacokinet. 27: 256-269 (1994).
D. R. Kohler & B. R. Goldspeil, "Evaluation of New Drugs: Paclitaxel
(Taxol)," Pharmacotherapy 14: 3-34 (1994).
J. L. Eiseman et al., "Plasma Pharmacokinetics and Tissue Distribution of
Paclitaxel in CD.sub.2 F.sub.1 Mice," Cancer Chemother. Pharmacol. 34:
465-471 (1994).
E. D. Harris, Jr., "Rheumatoid Arthritis: Pathophysiology and Implications
for Therapy," New Eng. J. Med. 322: 1277-1289 (1990).
D. Ingber et al., "Synthetic Analogues of Fumagillin that Inhibit
Angiogenesis and Suppress Tumor Growth," Nature 348: 555-557 (1990).
(Abstract) C. Tang et al., "Regression of Collagen-Induced Arthritis with
Taxol, a Microtubule Stabilizer," Arth. Rheum. 36 (Supplement): S45 (1993)
.
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Primary Examiner: Criares; Theodore J.
Attorney, Agent or Firm: Merchant, Gould, Smith, Edell, Welter & Schmidt
Goverment Interests
GOVERNMENT RIGHTS
This invention was supported by grants from the United States government,
namely Grant Nos. AR-38884, AR-36834, AR-40919, and AR-42200, from the
National Institutes of Health. Accordingly, the government may have
certain rights in this invention.
Claims
I claim:
1. A method for suppression of a progressive, inflammatory, autoimmune
arthritis in a mammal comprising administering to a mammal having or
susceptible to arthritis Taxol in a pharmacologically acceptable carrier
capable of solubilizing Taxol in a dose sufficient to suppress at least
one symptom of arthritis selected from the group of inflammation,
swelling, abnormal neovascularization, bone erosion, and cartilage
erosion.
2. The method of claim 1 wherein the mammal is a rat and the arthritis is
collagen-induced arthritis.
3. The method of claim 1 wherein the mammal is a human being and the
arthritis is rheumatoid arthritis.
4. The method of claim 1 wherein the pharmacologically acceptable carrier
is a 1: 1 dilution of ethanol and cremophor EL, further diluted with
saline.
5. The method of claim 1 wherein the dose sufficient to suppress at least
one symptom of arthritis is from about 7.5 mg/kg body weight to about 10
mg/kg body weight of Taxol.
6. The method of claim 1 wherein the dose sufficient to suppress at least
one symptom of arthritis is from about 0.075 mg/kg body weight to about
0.1 mg/kg body weight of Taxol.
7. The method of claim 1 wherein the dose sufficient to suppress at least
one symptom of arthritis is from about 0.75 mg/kg body weight to about 1.0
mg/kg body weight of Taxol.
8. The method of claim 1 further comprising administering to the mammal an
antiarthritic drug other than Taxol selected from the group consisting of
a nonsteroidal antiinflammatory agent, an organic gold derivative,
D-penicillamine, a 4-aminoquinoline, azathioprine, methotrexate,
cyclosporin, an angiogenesis inhibitor, a monoclonal antibody to T cells,
a monoclonal antibody to an adhesion molecule, and a monoclonal antibody
to a cytokine or growth factor.
9. The method of claim 8 wherein the antiarthritic drug other than Taxol is
the angiogenesis inhibitor O-(chloroacetylcarbamoyl) fumagillol.
10. A method for suppression of a progressive, inflammatory, autoimmune
arthritis in a mammal comprising:
(a) administering to a mammal having or susceptible to arthritis Taxol in a
pharmacologically acceptable carrier capable of solubilizing Taxol in a
dose sufficient to suppress at least one symptom of arthritis selected
from the group of inflammation, swelling, abnormal neovascularization,
bone erosion, and cartilage erosion; and
(b) administering to the mammal the angiogenesis inhibitor
O-(chloroacetylcarbamoyl) fumagillol in a dose sufficient to suppress at
least one symptom of arthritis;
the administration of both Taxol and ,AGM-1470 producing a greater degree
of suppression of at least one symptom of arthritis than does the
administration of the equivalent dose of either Taxol or
O-(chloroacetylcarbamoyl) fumagillol alone.
11. A method for suppression of a progressive, inflammatory, autoimmune
arthritis in a mammal comprising administering to a mammal having or
susceptible to arthritis a Taxol derivative in a pharmacologically
acceptable carrier capable of solubilizing the Taxol derivative in a dose
sufficient to suppress at least one symptom of arthritis selected from the
group of inflammation, swelling, abnormal neovascularization, bone
erosion, and cartilage erosion.
12. The method of claim 11 wherein the mammal is a rat and the arthritis is
collagen-induced arthritis.
13. The method of claim 11 wherein the mammal is a human being and the
arthritis is rheumatoid arthritis.
14. The method of claim 11 further comprising administering to the mammal
an antiarthritic drug other than the Taxol derivative selected from the
group consisting of a nonsteroidal antiinflammatory agent, an organic gold
derivative, D-penicillamine, a 4-aminoquinoline, azathioprine,
methotrexate, cyclosporin, an angiogenesis inhibitor, a monoclonal
antibody to T cells, a monoclonal antibody to an adhesion molecule, and a
monoclonal antibody to a cytokine or growth factor.
15. The method of claim 14 wherein the antiarthritic drug other than the
Taxol derivative is the angiogenesis inhibitor O-(chloroacetylcarbamoyl)
fumagillol.
Description
BACKGROUND OF THE INVENTION
This invention is directed to a method of suppression of a progressive,
inflammatory, autoimmune arthritis in a mammal, such as rheumatoid
arthritis. Despite advances in treatment, arthritis remains an extremely
serious health problem, particularly in view of the aging population in
the United States and other developed countries, because arthritis is
typically a disease of the elderly. Although rarely fatal, arthritis is a
major cause of morbidity, loss of time from work, lost productivity and
decrease in quality of life. It causes severe pair and loss of joint
mobility and can make doing even simple tasks difficult.
Among the most serious forms of arthritis is rheumatoid arthritis.
Rheumatoid arthritis is generally believed to be an autoimmune disease
that is believed to be associated with activity of autoreactive T cells.
It is believed that these cells cause the disease via a delayed-type
hypersensitivity reaction. Although it is not completely certain which
antigen these T cells recognize, one significant candidate is type II
collagen. The possibility exists that other antigens may also play a role
in the disease.
Substantial work has been done on genetic bases for susceptibility to the
disease. This work has focused on MHC haplotypes. Thus, it may be possible
to determine, from familial studies or direct genomic analysis, that some
individuals are at particular risk for the development of rheumatoid
arthritis.
Although a number of treatment methods and regimes exist for rheumatoid
arthritis, none of them is as yet completely satisfactory. These treatment
regimens include administration of non-steroidal anti-inflammatory drugs
such as acetylsalicylic acid (aspirin), ibuprofen, naproxen, and other
such agents, gold compounds, penicillamine, 4-aminoquinoline agents, and
immunomodulators.
Collagen-induced arthritis (CIA) is a T-cell dependent animal model of
rheumatoid arthritis (RA) (D. E. Trentham et al., "Autoimmunity to Type II
Collagen: An Experimental Model of Arthritis," J. Exp. Med. 146: 857-868
(1977)). Within two weeks after immunization with type II collagen (CII)
in IFA, susceptible rats develop polyarthritis with histologic changes of
pannus formation and bone/cartilage erosion. In addition, humoral and
cellular responses to CII occur in CIA as well as RA (E. Brahn, "Animal
Models of Rheumatoid Arthritis: Clues to Etiology and Treatment" in
Clinical Orthopedics and Related Research (B. Hahn, ed., Philadelphia, JB
Lippincott Company, 1991). Consequently, CIA is a useful animal model of
RA that serves as an in vivo system for the exploration of inflammatory
synovitis etiologies and for the investigation of potentially new
therapeutic interventions.
However, there exists a need for an improved treatment of rheumatoid
arthritis and other autoimmune forms of arthritis that can suppress or
ameliorate symptoms such as inflammation, swelling, abnormal
neovascularization, bone erosion, or cartilage erosion. Preferably, such
an improved method of treatment should be able to be combined with other
treatment methods, should work rapidly to cause regression or
stabilization of symptoms, and should be well tolerated. Preferably, such
a treatment regimen should also be adaptable to prophylaxis in susceptible
individuals.
SUMMARY
An improved method of suppression of a progressive, inflammatory,
autoimmune arthritis in a mammal involves the use of the drug Taxol. Taxol
is undergoing wide evaluation for treatment of malignancies, but has not
been evaluated or proposed for the treatment of arthritis.
In general, a method according to the present invention comprises
administering to a mammal having or susceptible to arthritis Taxol in a
pharmacologically acceptable carrier capable of solubilizing Taxol in a
dose sufficient to suppress at least one symptom of arthritis selected
from the group of inflammation, swelling, abnormal neovascularization,
bone erosion, and cartilage erosion.
The mammal can be a rat, in which case the arthritis can be
collagen-induced arthritis.
Alternatively, the mammal can be a human being, in which case the arthritis
can be rheumatoid arthritis.
Typically, the pharmacologically acceptable carrier is a 1: 1 dilution of
ethanol and cremophor EL, further diluted with saline.
Typically, the dose sufficient to suppress at least one symptom of
arthritis is from about 7.5 mg/kg body weight to about 10 mg/kg body
weight of Taxol. However, lower doses can be used, such as from about
0.075 mg/kg body weight to about 0.1 mg/kg body weight, or from about 0.75
mg/kg body weight to about 1 mg/kg body weight.
Alternatively, derivatives of Taxol substituted on the diterpene nucleus
can be used in place of Taxol.
Another aspect of the present invention is the use of Taxol in combination
with another antiarthritic drug. The antiarthritic drug other than Taxol
can be selected from the group consisting of a nonsteroidal
antiinflammatory agent, an organic gold derivative, D-penicillamine, a
4-aminoquinoline, azathioprine, methotrexate, cyclosporin, an angiogenesis
inhibitor, a monoclonal antibody to T cells, a monoclonal antibody to an
adhesion molecule, and a monoclonal antibody to a cytokine or growth
factor.
A preferred antiarthritic drug other than Taxol for combination therapy is
the angiogenesis inhibitor AGM-1470.
A preferred method for combination therapy comprises:
(1) administering to a mammal having or susceptible to arthritis Taxol in a
pharmacologically acceptable carrier capable of solubilizing Taxol in a
dose sufficient to suppress at least one symptom of arthritis selected
from the group of inflammation, swelling, abnormal neovascularization,
bone erosion, and cartilage erosion; and
(2) administering to the mammal the angiogenesis inhibitor AGM-1470 in a
dose sufficient to suppress at least one symptom of arthritis.
The administration of both Taxol and AGM-1470 produces a greater degree of
suppression of at least one symptom of arthritis than does the
administration of the equivalent dose of either Taxol or
O-(chloroacetylcarbamoyl) fumagillol (AGM-1470) alone
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention
will become better understood with reference to the following description,
appended claims, and accompanying drawings where:
FIG. 1 is the structural formula of Taxol;
FIG. 2 is a graph showing arthritis severity in rats as a function of the
number of days after immunization with type II collagen for rats left
untreated, rats treated with low-dose Taxol, and rats treated with
high-dose Taxol;
FIG. 3A is a photograph of a limb from a control group rat 28 days after
immunization with type II collagen (Example 1), showing swelling and other
changes characteristic of arthritis;
FIG. 3B is a radiograph of the limb shown in FIG. 3A, showing soft tissue
inflammation and bone destruction;
FIG. 3C is a photograph of a limb from a rat 28 days after immunization
with type II collagen treated with high-dose Taxol, showing no swelling or
other changes characteristic of arthritis;
FIG. 3D is a radiograph of the limb shown in FIG. 3C, showing the absence
of soft tissue inflammation and bone destruction, contrasted with FIG. 3B;
FIG. 4A is a photomicrograph of a synovial section from an arthritic
control group rat, demonstrating marked pannus, with bone and cartilage
erosion (P=pannus; C=cartilage; JS=joint space);
FIG. 4B is a photomicrograph of a synovial section from a rat treated with
high-dose Taxol, demonstrating minimal if any pannus, with preservation of
articular cartilage (C=cartilage; JS=joint space);
FIG. 5 is a graph showing arthritis severity in rats (Example 2) with
established arthritis treated With AGM-1470 (.box-solid.), Taxol
(.quadrature.), AGM-1470 in combination with Taxol (.diamond-solid.), and
controls (.diamond.);
FIG. 6A is a photograph of a hind limb from a control group rat 19 days
after arthritis onset (Example 2), showing swelling and other changes
characteristic of arthritis;
FIG. 6B is a radiograph of the hind limb shown in FIG. 6A, showing soft
tissue inflammation and bone destruction;
FIG. 6C is a photograph of a hind limb from a combination therapy group rat
(Example 2), showing regression of previously occurring swelling and other
changes characteristic of arthritis; and
FIG. 6D is a corresponding radiograph of the hind limb shown in FIG. 6C,
showing regression of soft tissue inflammation and bone destruction.
DESCRIPTION
I have developed a method for suppression of a progressive, inflammatory,
auto-immune arthritis in a mammal using the antineoplastic drug Taxol. As
used herein, the term "suppression" includes any or all of the following:
(1) amelioration of existing symptoms; (2) prevention of progression of
symptomatology in these progressive disease processes; (3) prevention of
the inception or occurrence of the disease in a susceptible subject, i.e.,
prophylaxis.
The method of treatment of the present invention uses Taxol, a microtubule
stabilizer. Taxol, whose structure is shown in FIG. 1, is an extremely
hydrophobic diterpene extracted from the dried inner stem bark of the
Western yew, Taxus brevifolia (M. C. Wani et al., "Plant Antitumor Agents
VI. The Isolation of Structure of Taxol, A Novel Antileukemic and
Antitumor Agent from Taxus Brevifolia," J. Am. Chem. Soc. 93: 2325-2327
(1971)) which grows primarily in old-growth forests of the Pacific
Northwest. The compound has a complex ring structure, with an ester side
chain at position C-13 that confers bioactivity in mammalian systems (S.
B. Horwitz, "Mechanism of Action of Taxol," Trans Pharmacol. Sci. 13:
134-136 (1992)) It has antineoplastic activity against a number of
neoplastic cell strains frequently used as models for Cancer (P. H.
Wiernik et al., "Phase One Clinical and Pharmacokinetic Study of Taxol,"
Cancer Res. 47: 2486-2493 (1987)). Attempts to synthesize the compound are
critical, since at present, the isolation of enough Taxol for one
patient-year of treatment requires more than sixty pounds of tree bark.
Taxol has now been partially synthesized from the needles of the European
Yew tree and more recently by a totally synthetic pathway (K. C. Nicolaou
et al., "Total Synthesis of Taxol," Nature 367: 630-634 (1994)).
Studies have demonstrated that Taxol has a unique mechanism of action.
Unlike other antimicrotubule agents that depolymerize microtubules, e.g.,
colchicine and vinblastine, Taxol has been shown to enhance microtubule
polymerization by preferentially binding to the .beta. subunit of tubulin
and shifting the dynamic equilibrium from tubulin dimers to stable
microtubule polymers, even in the absence of GTP and microtubule
associated proteins that are normally required (S. B. Horwitz (1992),
supra; P. B. Schiff & S. B. Horwitz, "Promotion of Microtubule Assembly in
Vitro by Taxol," Nature 277: 665-667 (1979)); N. Kumar, "Taxol-Induced
Polymerization of Purified Tubulin," J. Biol. Chem. 256: 10435-10441
(1981)). Because Taxol blocks reorganization of the microtubule
cytoskeleton (E. B. Horwitz (1992), supra; P. B. Schiff & S. B. Horwitz,
"Taxol Stabilizes Microtubules in Mouse Fibroblast Cells," Proc. Natl.
Acad. Sci. U.S.A. 77: 1561-1565 (1980)), it inhibits cell division at the
late G.sub.2 mitotic phase. This effect on mitotic spindles has been
suggested as the primary mechanism as an antitumor agent, although
microtubules are also important in many other functions including cell
migration, phagocytosis, chemotaxis, adhesion and intracellular transport
(E. B. Horwitz (1992), supra; R. L. Roberts et al., "Effects of Taxol on
Human Neutrophils," J. Immunol. 129: 2134-2141 (1982); A. Iannone et al.,
"Taxol Inhibits N-Formyl Methionyl-Leucyl-Phenylalanine (FMLP) Induced
Neutrophil Polarization and H.sub.2 O.sub.2 Production While Decreasing
[.sup.3 H]FMLP Binding," Agents Actions 21: 278-280 (1987); S. L. Newman
et al., "Differential Requirements for Cellular Cytoskeleton in Human
Macrophage Complement Receptor-Fc Receptor-Mediated Phagocytosis," J.
Immunol. 144: 967-974 (1991)). Recent studies also indicate that Taxol may
also have additional antineoplastic activity by up-regulating tumor
necrosis factor .alpha. (TNF .alpha.) and interleukin-1 (C. Bogdan & A.
Ding, "Taxol, a Microtubule-Stabilizing Antineoplastic Agent, Induces
Expression of Tumor Necrosis Factor a and Interleukin-1 in Macrophages,"
J. Leukoc. Biol. 52: 119-121 (1992); C. Bottex-Gauthier et al., "The
Effects of Taxol on the Macrophage Function: Interaction with Some
Immunological Parameters," Immunopharmacol. Immunotoxicol, 14: 39-61
(1992); C. L. Manthey et al., "Taxol Increases Steady-State Levels of
Lipopolysaccharide-Inducible Genes and Protein Tyrosine Phosphorylation in
Murine Macrophages," J. Immunol. 149: 2459-2465 (1992)).
Accordingly, a method of suppression of such autoimmune arthritis diseases
comprises administering to a mammal having or susceptible to arthritis
Taxol in a pharmacologically acceptable carrier capable of solubilizing
Taxol in a dose sufficient to suppress at least one symptom of arthritis
selected from the group of inflammation, swelling, abnormal
neovascularization, bone erosion, and cartilage erosion.
The mammal can be a rat, in which case the arthritis can be
collagen-induced arthritis, a well-recognized model for rheumatoid
arthritis in humans (see example 1).
Alternatively, the mammal can be a human being and the arthritis can be
rheumatoid arthritis.
The pharmacologically acceptable carrier is preferably a 1: 1 dilution of
ethanol and cremophor EL (Sigma Chemical Co. St. Louis, Mo.), further
diluted with saline. Other pharmacologically acceptable carriers capable
of solubilizing Taxol exist and are known in the art. Other agents, such
as buffers, stabilizers, and preservatives, can optionally be added as
needed.
In one alternative, the dose sufficient to suppress at least one symptom of
arthritis is from about 7.5 mg/kg body weight to about 10 mg/kg body
weight of Taxol. Typically, in this alternative, a dose of about 10 mg/kg
body weight is used, referred to as full-dose Taxol. However, without
being bound by this theory, Applicant believes that, in accord with the
experience gained with the use of other antineoplastic drugs in rheumatoid
arthritis, a dosage of one to two orders of magnitude less may be
appropriate in suppressing arthritis without leading to side effects. This
would lead to a dosage of as low as 0.1 mg/kg body weight. For example,
dosages of about 0.75 mg/kg body weight to about 1.0 mg/kg body weight or
of about 0.075 mg/kg body weight to about 0.1 mg/kg body weight can be
used. Of course, intermediate dosages can also be used. Even lower dosages
may be of benefit.
Typically, administration of Taxol occurs at 2-3 day intervals (see Example
1). However, again in accord with the experience gained with the use of
other antineoplastic drugs in rheumatoid arthritis, administration may
occur less frequently, e.g., weekly, biweekly, or even monthly.
Preferably, administration is intravenous or intraperitoneal. Other
injection routes can also be used, and are known in the art.
Alternatively, the drug can be administered orally.
The most effective mode of administration and dosage regimen for Taxol as
used in the methods of the present invention depend on the severity and
course of the disease, the patient's health, the response to treatment,
pharmacokinetic considerations such as the condition of the patient's
liver and/or kidneys that can affect the metabolism and/or excretion of
the administered Taxol, and the judgment of the treating physician.
Accordingly, the dosages should be titrated to the individual patient.
Another aspect of the present invention is a method of combination therapy
in which an antiarthritic drug other than Taxol is administered to the
mammal along with Taxol. This other antiarthritic drug can be any of the
following: (1) a nonsteroidal anti-inflammatory agent such as
acetylsalicylic acid (aspirin), ibuprofen, or naproxen; (2) an organic
gold derivative such as gold sodium thiomalate, aurothioglucose, or
auranofin; (3) D-penicillamine; (4) a 4-aminoquinoline agent such as
hydroxychloroquine; (5) azathioprine; (6) methotrexate; (7) cyclosporin;
(8) an angiogenesis inhibitor such as AGM-1470; (9) monoclonal antibodies
to T cells; (10) monoclonal antibodies to adhesion molecules and (11)
monoclonal antibodies to cytokines and growth factors.
A particularly preferred antiarthritic drug other than Taxol for
combination therapy is the angiogenesis inhibitor AGM-1470, as described
below in Example 2.
The dosage for the antiarthritic drug other than Taxol can be determined by
the treating physician in much the same way as the Taxol dose. A typical
high dose regimen for the preferred angiogenesis inhibitor AGM-1470 is
27.5 mg/kg, administered every other day, but, as with Taxol, this agent
can be administered at lower dosages and at a lower frequency.
When Taxol is administered in combination with another antiarthritic drug,
the administration of both Taxol and the other antiarthritic drug produces
a greater degree of suppression of at least one symptom of arthritis than
does the administration of the equivalent dose of either Taxol or the
other antiarthritic drug alone. This has been demonstrated for AGM-1470
(Example 2).
Alternatively, in place of Taxol, derivatives of Taxol can be used in a
method according to the present invention. Such derivatives can be
substituted on the diterpene nucleus, leaving the ester substituent at
C-13 which is significant for bioactivity, as discussed above. The
substituents can include lower alkyl (C.sub.1 -C.sub.5), halogen,
hydroxyl, and other groups. The side chains such as the ester groups and
the benzoyloxy group can also be substituted, such as on the methyl
moieties of the ester linkage or the aromatic moiety of the benzoyloxy
group. Such substituents may improve solubility or stability.
The present invention is illustrated by the following Examples. The Example
are for illustrative purposes only and are not intended to limit the
invention.
EXAMPLES
Regression of Collagen-Induced Arthritis in Rats by Treatment With Taxol
Materials and Methods
Animals
Syngeneic female Louvain (LOU) rats (120-150 g), housed in the UCLA
vivarium, were used in all experimental protocols.
Arthritis Induction
Rats, under ether anesthesia, were injected intradermally with 0.5 mg of
native chick collagen type II (CII) (Genzyme, Boston, Mass.) solubilized
in 0.1M acetic acid and emulsified in IFA (Difco, Detroit, Mich.) (D. E.
Trentham et al., "Autoimunity to Type II Collagen: An Experimental Model
of Arthritis," J. Exp. Med. 146: 857-868 (1977)). Between 90-100% of rats
typically develop synovitis by day 9 post-immunization.
Experimental Design
Taxol (Sigma, St. Louis, Mo.) was initially solubilized in a 1: 1 dilution
of ethanol and cremophor EL (Sigma, St. Louis, Mo.). Normal saline was
added to make a final concentration of 4.8 mg/ml Taxol in 5% w/v ethanol
and 5% w/v cremophor EL prior to administration by i.p. injection. A total
of 45 rats in four protocols were used: a control group that received
vehicle alone, and three Taxol treatment groups consisting of a prevention
and two suppression protocols. In the prevention protocol, 1 mg/kg body
weight of Taxol was administered on day 2 after CII immunization in an
attempt to preclude CIA induction. Five subsequent doses were given on
days 5, 7, 9, 12, and 14. In the two suppression protocols, rats were
allowed to develop arthritis and Taxol was then given on alternate days
beginning on the day of arthritis onset (day 9). In the "high dose"
suppression protocol, full dose (1 mg/kg body weight) Taxol was used until
day 19 when it was discontinued. In the "low dose" suppression protocol,
because of concerns about potential toxicity with the higher dosage
schedule, Taxol (10 mg/kg body weight) was administered on days 9, 11, 13
and then 75% of this dose (7.5 mg/kg body weight) was continued on
alternate days through day 21. The dose administered on day 21 was added
in an attempt to extend the benefits of Taxol since preliminary data
indicated that recrudescence of arthritis could occur after
discontinuation of Taxol. Final body weights were determined on day 28 at
the completion of the study.
Arthritis Assessment
The incidence and severity of the arthritis were both evaluated. The
incidence was determined by the number of rats with clinical evidence of
joint inflammation during the study period. The severity of inflammation
of each paw was evaluated daily by the same investigator using an integer
scale that ranged from 0 to 4. This quantification method scale was based
on increasing standardized levels of swelling and periarticular erythema
with 0 being normal and 4 being severe. Scores were independently
confirmed by other investigators throughout the study with excellent
inter-observer agreement. The sum of the scores for all four limbs
(maximum score=16) is the arthritic index for each rat (D. E. Trentham
(1977), supra; R. T. Schoen et al., "Antigen-Specific Suppression of Type
II Collagen-Induced Arthritis by Collagen-Coupled Spleen Cells," J.
Immunol. 128: 717-719 (1982); D. E. Trentham & C. E. Brinckerhoff,
"Augmentation of Collagen Arthritis by Synthetic Analogues of Retinoic
Acid," J. Immunol. 129: 2668-2672 (1982)). An index score between 6 and 8
is considered severe disease since CIA usually affects only hind limbs.
Photographs of selected limbs were obtained on day 28.
Radiographic scores, determined by an investigator blinded to treatment
intervention, were determined for the hind limbs on day 28 by the extent
of soft tissue swelling, joint space narrowing, bone destruction, and
periosteal new bone formation (E. Brahn & D. E. Trentham, "Antigen
Specific Suppression of Collagen Arthritis by Adoptive Transfer of Spleen
Cells," Clin. Immunol. Immunopathol. 31: 124 (1984)). An integer scale
from 0 to 3 was used to quantify each limb (0=normal, 1=soft tissue
swelling, 2=early erosions of bone, 3=severe bone destruction and/or
ankylosis). The scores were assigned by a blinded investigator. The sum of
both hind limb scores for each rat represented the radiographic joint
index (maximum score per rat=6) (D. E. Trentham & C. E. Brinckerhoff
(1982), supra; E. Brahn & D. E. Trentham (1984), supra).
Histologic Analysis
Hind limbs were randomly selected on day 28 from the control and Taxol
treated groups for histologic analysis by a pathologist blinded to
treatment protocols. Thymuses and spleens were also harvested for weight
determination and histologic evaluation. Hematoxylin and eosin stained
sections were reviewed by a blinded pathologist using light microscopy.
Immune Responses
Delayed type hypersensitivity (DTH) to CII was determined by a radiometric
ear assay completed on day 28 (D. E. Trentham & C. E. Brinckerhoff (1982),
supra; W. J. McCune et al., "Gold Does Not Alter the Arthritic, Humoral,
or Cellular Responses in Rats with Type II Collagen-Induced Arthritis,"
Arthritis Rheum. 23: 932-936 (1980)). Based on previous studies,
radiometric ear indices.gtoreq.1.4 represent a significant response to
CII. The presence of anti-CII IgG antibodies was determined by an
enzyme-linked immunosorbent assay (ELISA) (D. E. Trentham & R. A.
Dynesius-Trentham, "Attenuation of an Adjuvant Arthritis by Type II
Collagen," J. Immunol. 130: 2689-2692 (1983); G. Ku et al., "Prevention of
Experimental Autoimmune Arthritis with a Peptide Fragment of Type II
Collagen," Eur. J. Immunol. 23: 591-599 (1993); D. Peacock et al.,
"Angiogenesis Inhibition Suppresses Collagen Arthritis," J. Exp. Med. 175:
1135-1138 (1992)). Serum samples were obtained on day 26, diluted to 1:
2560, and the results were expressed as the mean optical density at 490 nm
of quadruplicate aliquots. The 1: 2560 dilution was chosen because it
represents the mid-linear portion of a standardized ELISA curve for pooled
arthritic rat serum that was evaluated concurrently with the experimental
serum. Normal rat serum backgrounds at this dilution are zero and readily
distinguishable from collagen-immunized rat serum.
Statistics
Students' t-test was used to evaluate the group means of the continuous
variables. The Chi square test was used to evaluate the proportionate
group frequencies of dichotomous variables. Yeats' correction was used
where indicated and the results were defined as significant at the P<0.05
level.
Results
Clinical Evaluations
None of the rats in the Taxol prevention protocol developed arthritis (even
after discontinuation of Taxol) compared to 94% in the vehicle control
group (Table 1). Since all rats were required to have arthritis prior to
entry into either of the two Taxol suppression protocols, the incidence of
arthritis was 100% in these groups. Within 5 days of Taxol institution,
both high and low dose protocols demonstrated clear reductions in
arthritis severity compared to controls (FIGS. 2 and 3). The study
protocol required that all rats in the low-dose and high-dose Taxol
arthritis suppression protocols (Taxol Low and Taxol High in FIG. 2) have
arthritis prior to the institution of taxol. None of the rats in the taxol
arthritis prevention protocol developed arthritis. Arthritis severity
nadirs occurred by day 21 (P<0.0000001 and P<0.0001 for the high and low
dose groups, respectively). These rats were observed for an additional
week after discontinuation of Taxol and, within the first 3 or 4 days of
this wash out period, a return of soft tissue swelling was evident in some
rats.
TABLE 1
______________________________________
Clinical and Radiographic Effects of Taxol
on Collagen-Induced Arthritis
Taxol-
treated
rats, Taxol-treated rats.
Control
prevention
suppression protocol
rats protocol High-dose Low-dose
______________________________________
No. of rats
17 8 10 10
Clinical 94 0* 100.dagger.
100.dagger.
incidence
of arthritis, %
Clinical 5.9 .+-. 0.5
0.dagger-dbl.
0.7 .+-. 0.3.dagger-dbl.
2.0 .+-. 0.5*
severity
of arthritis
(day 21),
mean .+-. SEM
Radiographic
4.0 .+-. 0.4
0.sctn. 1.2 .+-. 0.2*
1.3 .+-. 0.2.paragraph.
score
(day 28),
mean .+-. SEM
Radiographic
94# 0* 0** 20.paragraph.
erosions
(day 28), %
______________________________________
*P < 0.0001 compared with controls.
.dagger.The taxol suppression protocols required that the rats have
arthritis prior to therapeutic intervention.
.dagger-dbl.P < 0.0000001 compared with controls.
.sctn.P < 0.000001 compared with controls.
.paragraph.P < 0.001 compared with controls.
#All 16 rats in this group that had clinical arthritis also had erosions.
**P < 0.00001 compared with controls.
Rats that tolerated their Taxol therapy had stable weights throughout the
study that were comparable to higher, but not significantly different,
than the control group. No deaths were noted in the prevention protocol,
possibly because they received the fewest Taxol doses and at no point
developed the intercurrent illness of CIA. Three rats in the more
protracted low dose regimen and one rat in the high dose protocol
developed late onset weight loss, diarrhea, and subsequently died (median
death at 25 days post CII immunization). Both of these sequelae have been
described in humans administered Taxol for advanced cancer therapy. All
other rats, aside from the development of arthritis, appeared healthy.
Peripheral blood hematocrits and white blood cell counts were similar in
all groups although rats intolerant of Taxol had a selective neutropenia
(another recognized consequence observed in human studies) that may
predispose them to infectious complications.
Radiographic Scores
Hind limb radiographs were obtained and blindly read on day 28. None of the
rats in the Taxol prevention protocol (which also lacked any clinical
arthritis) manifested any radiographic changes compared to 94% in the
control group (Table 1). Since rats in either of the Taxol suppression
protocol groups had been off therapy for at least a week by day 28, their
radiographic index primarily reflects the recrudescence of soft tissue
swelling during this interval. Both the high and low dose Taxol groups had
significantly less disease (FIG. 3C and 3D, Table 1) compared to controls.
Only 2 of the 28 total Taxol recipients (7%) demonstrated any evidence of
even minimal erosions (all in the low dose suppression group) compared to
94% with erosions in the control group. All of the 16 control rats with
clinical arthritis had significant erosions (100%) and the single rat
without clinical arthritis also lacked radiographic changes.
Synovial Pathology
Synovial sections obtained from control group rats demonstrated marked
pannus with bone and cartilage erosions (FIG. 4A; P=pannus, C=cartilage,
J=joint space). In general, Taxol treated rats had minimal if any pannus
with preservation of articular cartilage (FIG. 4B; C=cartilage, J=joint
space).
Immunologic Effects
The mean delayed-type hypersensitivity (DTH) responses in the Taxol treated
groups were not significantly different from the control group (Table 2).
The mean IgG anti-CII antibody titer as measured by ELISA, however, was
significantly higher in the control group compared to the prevention and
suppression groups (Table 2). Blinded histologic evaluation of thymuses
and spleens showed no overt histologic changes compared to controls.
Spleen weights, however, were higher in the Taxol treated rats (392
mg.+-.18 vs 299.+-.44, P<0.05). Taxol recipients tended to weigh more than
the severely arthritic controls. If the spleen sizes were compared as a
percentage of body weight, there were no significant differences.
TABLE 2
__________________________________________________________________________
Immunologic Effects of Taxol on Collagen-Induced Arthritis
Taxol-treated
Taxol-treated rats,
rats, prevention
suppression protocol
Control rats
protocol
High-dose
Low-dose
__________________________________________________________________________
IgG anti-collagen antibodies.dagger.
0.17 .+-. 0.002
0.09 .+-. 0.007.dagger-dbl.
0.13 .+-. 0.007.dagger-dbl.
0.16 .+-. 0.002.dagger-dbl.
Delayed-type hypersensitivity
3.06 .+-. 0.19
2.98 .+-. 0.25
3.09 .+-. 0.29
ND
__________________________________________________________________________
*Assays were performed at day 28. Values are the mean .+-. SEM. ND = not
done.
.dagger.Optical density at 490 nm, 1:2,560 dilution.
.dagger-dbl.P < 0.01 compared with controls.
This is the first study to evaluate Taxol in an autoimmune disease. The
experiments demonstrate that Taxol could completely abrogate arthritis
onset if initiated 2 days after CII immunization. There was a marked
reduction in CIA severity after 3 injections of Taxol for established
arthritis and the "high dose" protocol was more effective than the "low
dose" protocol. The severity of arthritis continued to decrease throughout
the duration of Taxol administration but began to rise within four days
after the cessation of treatment in both suppression protocols. This
suggests that chronic intermittent therapy is needed to control
established CIA, possibly because in the absence of Taxol, which has a
rapid systemic clearance, microtubule stabilization is not static and is
in constant equilibrium with the .alpha. and .beta. tubulin dimers. In
contrast, early intervention with Taxol (prevention protocol) appeared to
attenuate the need for continuous therapy. This might be due to Taxol's
inhibition of CII phagocytosis and/or processing to a recently identified
pathogenic 12 mer epitope (G. Ku et al. (1993), supra), both microtubule
dependent steps. The impact of Taxol on CIA is relatively rapid since
permanent joint damage, as defined by radiographically evident erosions,
was precluded.
Because microtubule function is important in multiple physiologic
processes, further studies are needed to clarify the critical immunologic
and inflammatory effects of Taxol therapy on autoimmune mechanisms.
Although overall DTH responses to whole CII were not significantly
altered, prior studies in CIA indicate that afferent sensitization to 99%
of the parent molecule, excluding the pathogenic epitope at position 62-73
on cyanogen bromide fragment 11, are not predictive of disease
susceptibility in Louvain rats (G. Ku et al. (1993), supra). Only a
limited repertoire of CD4.sup.+ T cell clones, that recognize this
processed sequence, are necessary for CIA induction (D. Peacock et al.
(1992), supra). Although antibodies to CII were statistically lower in
Taxol recipients, the biological significance of this finding is unclear
since all rats produced high titer anti-CII IgG antibodies. In addition to
its inhibition of cell mitosis, migration, chemotaxis, and adhesion, Taxol
can also suppress intracellular transport and neutrophil H.sub.2 O.sub.2
production (E. Rowinsky et al., "Taxol: A Novel Investigational
Antimicrotubule Agent,." J. Nat. Cancer Inst. 82: 1247-1257 (1990)). All
these important components of the inflammatory response are linked to
microtubule function. Taxol has recently been shown (C. Bottex-Gauthier et
al. (1992), supra; C. L. Manthey et al. (1992), supra) to have complex
effects on murine macrophage expression of TNF-.alpha. and
TNF-.alpha.-receptors by rapid induction of tyrosine phosphorylation of a
41- and 42 -kDa protein, a mechanism previously demonstrated with LPS. In
macrophages exposed to Taxol, cell cycle-independent TNF-.alpha.
transcription was upregulated but TNF-.alpha. receptors were actively
internalized. This might be an important mechanism given the critical role
TNF-.alpha. plays in CIA (E. Brahn et al., "Effects of Tumor Necrosis
Factor Alpha (TNF-.alpha.) on Collagen Arthritis," Lymphokine Cytokine
Res. 11: 253-256 (1992)).,
In addition to its potential as a therapeutic agent, Taxol represents an
important tool to study microtubule-dependent cellular events. It binds
specifically to cells with a high affinity that is reversed with
colchicine or vinblastine, agents that depolymerize microtubules (S. B.
Horwitz (1992), supra). Taxol has already been used to isolate tubulin and
microtubule-associated proteins from a variety of cells (S. B. Horwitz
(1992), supra). Mutant tumors have also been developed that are either
Taxol-resistant (because of aberrant tubulin subunits) or Taxol-dependent
(for cell replication).
Taxol is a prototypic compound for a new class of potential antirheumatic
agents. The study reported in this Example demonstrates that Taxol is a
disease modifying drug in rat CIA. It could also be considered a
candidate, because of its unique mechanism of action, for combination
therapies (see Example 2). Further investigations of Taxol in CIA and
other autoimmune diseases appear to be warranted.
EXAMPLE 2
Regression of Collagen-Induced Arthritis in Rats by Treatment With Taxol
and an Angiogenesis Inhibitor
As a refinement of the studies reported in Example 1, regression of
collagen-induced arthritis in rats by treatment with Taxol and an
angiogenesis inhibitor was studied.
Collagen induced arthritis (CIA) in rats is a T-cell dependent animal model
of chronic inflammatory arthritis resulting in pannus and joint
destruction similar to rheumatoid arthritis (RA) (D. E. Trentham (1977),
supra). CIA is induced by intradermal injection of native collagen type II
(CII) emulsified in incomplete Freund's adjuvant with reliable development
of polyarthritis in greater than 90% of recipients 10 to 12 days later.
Within 4 weeks of CIA induction, fulminant synovitis results in extensive
erosions and frank ankylosis of involved joints. In addition, rats
immunized with CII develop antibodies and delayed-type hypersensitivity
(DTH) to the antigen (E. Brahn (1991), supra).
In adults, physiologic angiogenesis is usually limited to wound healing and
reproductive functions. Abnormal neovascularization, however, contributes
to pathologic states, including tumor growth, proliferative retinopathy,
and inflammatory synovitis (J. Folkman & M. Klagsbrun, Science, 235: 442
(1987); E. S. Kimball & J. L. Gross, Agents Actions, 34: 329 (1991); D. J.
Peacock et al., J. Exp. Med. 175: 1135 (1992); D. J. Peacock et al.,
Arthritis Rheum. 35: S51 (1992) (abstract); D. J. Peacock et al.,
Arthritis Rheum. 35: S140 (1992) (abstract)). A potent inhibitor of
angiogenesis, AGM-1470 is a synthetic derivative of fumagillin, a
naturally occurring product of Aspergillus fumigatus fresenius (D. Ingber
et al., Nature 348: 555 (1990). AGM-1470 has demonstrated in vivo efficacy
against solid tumors in animal models (D. Ingber et al. (1990), supra; H.
Brem & J. Folkman, J. Ped. Surg. 28: 445 (1993) and in vitro benefits in
human tumor studies (Y. Takamiya et al., J. Neurosurg. 78: 470 (1993). It
is currently undergoing phase I trials in patients with Kaposi's Sarcoma
and AIDS. In adjuvant and CIA rat models of synovitis, AGM-1470 has been
shown to effectively prevent pannus formation if given prior to arthritis
onset and to partially regress established arthritis (D. J. Peacock et
al., J. Exp. Med. 175: 1135 (1992); D. J. Peacock et al., Arthritis Rheum.
35: S51 (1992) (abstract)).
The properties of Taxol were discussed above, with respect to its
antineoplastic and antirheumatic action.
Standard anti-rheumatic therapy has typically involved the trial of
sequential single therapeutic drugs in an effort to control progressive
disease. Recent interest has focused on combination therapy where two or
more agents are used to potentially increase overall efficacy and to
reduce the incidence of adverse effects. Beneficial results with
traditional disease modifying anti-rheumatic drugs (DMARDs) in combination
have been difficult to demonstrate in humans (H. E. Paulus, Sem. Arthritis
Rheum. 23 (Suppl. 1): 19 (1993)). AGM-1470 and Taxol, which represent two
new classes of agents with specific mechanisms of action, were chosen as
ideal candidates for concurrent use in an effort to increase their already
considerable individual effectiveness in the CIA animal system.
Materials and Methods
Experimental Design
Syngeneic 8 week old female Louvain (LOU) rats were used in all
experimental protocols. Under ether anesthesia, rats were immunized
intradermally on day 0 with 0.5 mg of native chick CII (Genzyme, Boston,
Mass.) solubilized in 0.1M acetic acid and emulsified in IFA (Difco,
Detroit, Mich.) (D. E. Trentham et al. (1977), supra). Synovitis typically
develops in the hind legs of 90-100% of rats within 10-12 days. 100% of
rats by definition had arthritis entering the study. Four protocols (total
n=61) consisted of a control group given no active agent (n=12), or
AGM-1470 (n=14) and Taxol (n=11) administered as single agents, or in
combination (n=24). AGM-1470 was solubilized in normal saline, with 10%
ethanol and 5% gum arabic, and administered on alternate days by
subcutaneous injection of 0.3 ml containing a dose of 27.5 mg/kg. Taxol
(Sigma, St. Louis, Mo.) was solubilized in a 1: 1 ethanol/cremophor EL
(Sigma, St. Louis, Mo.) solution. Normal saline was added to make a final
concentration of 2.4 mg/ml Taxol in 5% ethanol and 5% cremophor prior to
j.p. injection. The dosing schedule of single agent Taxol was on alternate
days and consisted of 0.5 ml/rat (10 mg/kg Taxol) for the first 3 doses,
followed by a 25% reduced maintenance dosage of 7.5 mg/kg thereafter. All
treatment protocols began on the day of arthritis onset and continued for
the duration of the 29 day study. An initial pilot study of rats given
combination AGM-1470 and Taxol therapy demonstrated rapid and near total
suppression of arthritis, but with a high mortality within 11 days of its
initiation. This indicated that a reduction in the Taxol dose, when used
concurrently with AGM-1470, was necessary. The resulting combination
protocol with AGM-1470 employed Taxol at 7.5 mg/kg for all doses.
Arthritis Assessment
The degree of arthritis severity was recorded by daily scoring of each paw.
An integer scale of 0-4 was used to quantify the level of erythema and
swelling with 0=normal and 4=maximum. The sum of all four paws (a maximum
score of 16 per rat) represented the arthritis index (D. E. Trentham
(1977), supra). Because CIA typically affects only the hind legs, a score
of 6 to 8 reflects severe arthritis. Photographs of selected limbs were
obtained on day 29.
Radiologic scoring of hind limbs was determined by an investigator, blinded
to treatment protocol, on day 29 using a scoring system of 0-3, based on
the degree of soft tissue swelling, joint space narrowing, periosteal new
bone formation, and the presence of erosions and/or ankylosis (0=normal,
3=maximum joint destruction) (E. Brahn & D. E. Trentham, Clin. Immunol.
Immunopathol. 31: 124 (1984)). The radiographic joint index represented
the sum of both hind legs, with a maximum possible score of 6.
Cellular and Humoral Immunity
An in vivo radiometric ear assay completed on day 28 was used to measure
DTH to CII (D. E. Trentham & C. E. Brinckerhoff, J. Immunol. 129: 2668
(1982)). A radiometric ear index.gtoreq.1.4 is considered a significant
response to CII, based on previous studies. IgG antibodies to CII were
measured in quadruplicate aliquots from serum obtained on day 19 post
arthritis onset using an enzyme-linked immunosorbent assay (ELISA) (E.
Brahn & D. E. Trentham, Cell. Immunol. 86: 421 (1984); E. Brahn & D. E.
Trentham, Cell. Immunol. 118: 491 (1989)). Antibody titers were expressed
as the absorbance at 490 nm of a 1: 2560 dilution of serum and normalized
against a previously standardized curve.
Statistical Analysis
Student's t-test was used to analyze group means of continuous variables.
Results were considered significant at p<0.05.
Results
A significant reduction in arthritis severity (p<0.05) was evident in rats
treated with single agent AGM-1470 or Taxol, compared to controls rats,
within 9 and 11 days of arthritis onset, respectively (FIG. 5; AGM-1470
(.box-solid.), Taxol (.quadrature.), AGM-1470 in combination with Taxol
(.diamond-solid.), and controls (.diamond.)) Compared to controls, rats
treated with single agent AGM-1470 or Taxol had a significant reduction in
clinical arthritis by Days 9 and 11 postarthritis onset, respectively
(P<0.05). Combination therapy resulted in significant arthritis
suppression, compared to control animals, by Day 4 (P<0.05) that continued
throughout the study to Day 19 (P<0.00001).
The efficacy of single agent AGM-1470 or Taxol was comparable at all time
points during the treatment period. There were no treatment related deaths
or episodes of diarrhea in the single agent studies. Although there were
no treatment related deaths in the rats receiving combination therapy with
the reduced Taxol dose of 7.5 mg/kg, some rats, however, developed
transient weight loss and diarrhea, and both agents were temporarily
suspended between days 6 and 13 in the combination group protocol. The
affected rats demonstrated reversible granulocytopenia. Within 4 days of
combination therapy initiation, a significant reduction of arthritis
severity compared to controls (p<0.05) was noted and maintained throughout
the remainder of the study period (p<0.00001 by day 19 post arthritis
onset) (FIG. 5). Combination therapy was significantly better than single
agent AGM-1470 or Taxol by day 5 and 6 post onset of arthritis,
respectively. The maximum mean arthritis score of rats in the combination
protocol (Table 3) was 2.8 and occurred on day 3 post arthritis onset.
Maximum mean arthritis scores of 4.2 and 3.8 were attained by Taxol and
AGM-1470 treated rats, respectively, on day 5 post arthritis onset,
compared to control rats whose arthritis continued to increase throughout
the study to a maximum score of 5.4 on day 19 post arthritis onset (FIG.
5). Selected hind limbs of control and combination protocol rats at the
termination of the 29 day study are depicted in FIGS. 6A-6D. Marked
clinical inflammation and bone destruction were found in the control group
and this was absent in the combination therapy group. Blinded radiographic
scores of the hind limbs of combination and AGM-1470 treated rats were
significantly lower than controls (Table 3). Radiographic scores of
combination therapy rats were significantly lower than the single agent
Taxol treated rats.
Clinical and radiographic findings on Day 19 postarthritis onset are shown
in FIGS. 6A-6D. FIGS. 6A and 6B show a representative hind limb and
corresponding radiograph from a control group rat. Note the marked soft
tissue inflammation and joint destruction. FIGS. 6C and 6D show a
representative hind limb and corresponding radiograph from a combination
therapy group rat. This limb had an earlier maximum clinical arthritis
severity score of 3 on Day 3 postarthritis onset. With continued therapy,
it regressed to a clinical and blinded radiographic score of 0 (i.e., no
evidence of arthritis).
TABLE 3
__________________________________________________________________________
Arthritic and Immunologic Assessments in CIA (Example 2)
Maximum arthritis
Radiographic
Group Arthritis index.sup.a
score index.sup.b
Antibody to CII.sup.c
DTH to CII.sup.d
__________________________________________________________________________
Control
5.40 .+-. 0.98
5.40 .+-. 0.98
4.30 .+-. 0.72
0.14 .+-. 0.009
2.84 .+-. 0.40
AGM-1470
2.36 .+-. 0.59.sup.e
4.14 .+-. 0.52
1.58 .+-. 0.34.sup.f
0.12 .+-. 0.011
4.24 .+-. 0.45
Taxol 2.00 .+-. 0.63.sup.e
3.81 .+-. 0.60
2.27 .+-. 0.71
0.11 .+-. 0.005.sup.g
3.49 .+-. 0.66
Combination
0.82 .+-. 0.27.sup.h
2.76 .+-. 0.33.sup.f,i
0.94 .+-. 0.29.sup.j
0.11 .+-. 0.008.sup.e
3.28 .+-. 0.32
__________________________________________________________________________
.sup.a Mean arthritis index on Day 19 postarthritis onset .+-. SEM.
.sup.b Mean radiographic index on Day 19 postarthritis onset.
.sup.c Mean OD at 490 nm of a 1:2560 dilution.
.sup.d Mean radiometric ear index.
.sup.e P < 0.05 compared to controls.
.sup.f P < 0.005 compared to controls.
.sup.g P < 0.01 compared to controls.
.sup.h P < 0.00001 compared to controls; P < 0.05 compared to single agen
Taxol and AGM1470.
.sup.i P < 0.05 compared to AGM1470.
.sup.j P < 0.0001 compared to control group; P < 0.05 compared to Taxol
group.
Sensitization to CII, as measured by DTH and IgG anti-CII antibodies, was
evident in all treated and control groups by day 19 after arthritis onset.
However, the mean anti-CII antibody titer was significantly higher in the
control groups compared with the Taxol (p<0.01) and combination therapy
(p<0.05) groups. DTH levels were comparable within all treatment groups
and control rats.
In the study reported in this Example, the concurrent administration of
AGM-1470 and Taxol resulted in significant reduction of arthritis severity
as judged by both clinical and radiographic criteria. Suppression of
established arthritis was studied because either agent alone, if given
early prior to clinical arthritis, will prevent CIA. Prevention of CIA has
always been easier to achieve than regressing existing disease and may not
be a clinically relevant comparison for progressive human synovitis
conditions such as rheumatoid arthritis. The therapeutic benefit of
AGM-1470 and Taxol in combination was better clinically and
radiographically compared to either agent alone. The magnitude of
differences in radiographic scores was less than the clinical scores. This
may reflect the nonquantitative aspect of the radiographic scoring system
for the degree of soft tissue swelling, resulting in less sensitivity at
milder disease levels. Previous work has demonstrated that AGM-1470
functions as an angiogenesis inhibitor in CIA and adjuvant arthritis
models and not as an immunosuppressive agent (D. J. Peacock et al., J.
Exp. Med. 175: 1135 (1992); D. J. Peacock et al., Arthritis Rheum. 35: S51
(1992) (abstract))).
Conditions manifesting pannus require neovascularization to occur and to be
maintained. AGM-1470 is one of the few angiogenesis inhibitors to be
evaluated in vivo. It has demonstrated efficacy in angiogenesis inhibition
and suppression of solid tumor growth in a variety of cancer models (D.
Ingber et al. (1990), supra; H. Brem & J. Folkman (1993), supra; Y.
Takamiya et al. (1993), supra). It has little effect on nonreplicating
endothelial cells and angio-independent ascitic leukemic cells, suggesting
that it is not merely an antiproliferative agent (D. Ingber et al. (1990),
supra). Suppression of vasculitis in a murine model of Kawasaki's Disease
has also been demonstrated using AGM-1470 (D. J. Peacock et al., Arthritis
Rheum. 36: S93 (1993) (abstract)). The primary mechanisms of action of
AGM-1470 are currently under investigation. In vitro, AGM-1470 inhibits
fibroblast growth factor (FGF) induced stimulation of endothelial cell
migration, proliferation, and capillary tube formation (D. Ingber et al.
(1990), supra; H. Brem et al., Surg. Forum 42: 439 (1991); M. Kusaka et
al. Biochem. Biophys. Res. Comm. 174: 1070 (1991)), all key steps in the
process of neovascularization (D. C. Billington, Drug Des. Dis. 8: 3
(1991)). AGM-1470 also inhibits in vivo neovascularization induced by
basic FGF in the cornea micropocket model (E. M. Gonzalez et al., Invest.
Ophthamol. Vis. Sci. 33: 777 (1992) (abstract)). Possible additional
mechanisms of angioinhibition may include stimulation or inhibition of
other cytokines associated with angiogenesis regulation such as
TNF-.alpha., ECGF, IFN-.alpha. and IFN-.gamma. (J. Folkman & M. Klagsbrun
(1987), supra; N. Sato et al., J. Invest. Dermatol. 95 (Suppl. 6): 85S
(1990)). Vascular effects may also occur through various endogenous
inhibitors such as TIMP-1 and TIMP-2, which regulate the activity of
matrix metalloproteinases required for penetration of the basement
membrane by activated endothelial cells during neovascularization (M. A.
Moses et al., Science 248: 1408 (1990); M. A. Moses & R. Langer J. Cell
Biochem. 47: 230 (1991)). Modest angiogenesis inhibitory activity has been
associated with several agents used in autoimmune therapies. Methotrexate,
cyclophosphamide, and azathioprine have a direct inhibitory effect on many
cell types undergoing rapid turnover although of these, only methotrexate
has been shown to be angiostatic in vitro and in vivo (S. Hirata et al.,
Arthritis Rheum. 32: 1065 (1989)). D-penicillamine, another anti-rheumatic
drug, has also demonstrated an angioinhibitory capacity (T. Matsubara et
al., J. Clin. Invest. 83: 158 (1989)). In addition, the thiol moiety of
gold compounds has inhibitory effects upon macrophage mediated angiogenic
activity (A. E. Koch et al., Agents Actions 34: 350 (1991)). Finally,
chloroquine and the sulfasalazine metabolite, sulfapyridine, have both
been ascribed angiogenesis inhibitory properties (A. L. Inyang et al.,
Cell Biol. Int. Rep. 14: 35 (1990); R. Madhok et al., J. Rheum. 18: 199
(1991)). Despite these findings, other physiologic actions characteristic
of these anti-rheumatic agents make it difficult to determine the
importance of anglogenesis inhibition in their activity in autoimmune
therapy.
Taxol, with its unique action on the microtubule system, has both
immunologic and anti-inflammatory effects on autoimmune mechanisms (E. K.
Rowinsky et al. (1990), supra). In vitro studies have suggested that Taxol
acts as a phase-specific anti-cancer agent that is particularly cytotoxic
for cells undergoing mitosis (N. M. Lopes et al., Cancer Chemother.
Pharmacol. 32: 235 (1993)). A potent inhibitor of eukaryotic cell
replication, Taxol blocks cells in the late G2 mitotic phase (S. B.
Horwitz (1992), supra). Human leukemic cells exposed to Taxol in vitro
display bundles of disorganized microtubules and abnormal spindle aster
formations (P. Schiff & S. B. Horwitz, Proc. Natl. Acad. Sci. (USA) 77:
1561 (1980)).
The mechanism of arthritis suppression with Taxol may be multifold. Rapidly
proliferating inflammatory pannus cells may be more susceptible to Taxol's
phase specific cytotoxic effects. By interfering with normal microtubule
function, Taxol inhibits cell mitosis, migration, chemotaxis,
intracellular transport, and neutrophil H.sub.2 O.sub.2 production (E. K.
Rowinsky et al. (1990), supra). Suppression of intracellular transport by
Taxol may also interfere with macrophage processing and presentation of
CII, since it can permanently block the induction of CIA if given early
(see Example 1). Because microtubules are critical components of
coordinated endothelial cell migration (D. S. Ettenson & A. I. Gotlieb,
Lab. Invest. 66: 722 (1992); S. R. Gordon & C. Staley, Cell Motil.
Cytoskel. 16: 47 (1990)), one of the key steps in neovascularization,
Taxol could have anti-angiogenic activity. Strong immune responses to CII,
as measured by DTH and antibody assays, suggest that immunosuppression was
not a major factor in determining therapeutic outcome in this
antigen-specific, T-cell-mediated autoimmune animal model. Although
antibodies to CII were statistically lower in the Taxol treated rats, the
significance of this is unclear since all rats produced high titer
anti-CII IgG antibodies (Table 3).
Taxol's effect on an antigen-specific humoral immune response has not been
previously reported. It has been linked to inhibition of several cell
mediated processes including lymphocyte proliferation (J. A. Cuthbert & J.
Shay, J. Cell Physiol. 116: 127 (1983) and selective neutrophil/macrophage
functions (R. L. Roberts et al., J. Immunol. 129: 2134 (1982)). By a
mechanism previously described with lipopolysaccharide (LPS), Taxol
upregulates TNF-.alpha. expression but down-regulates TNF-.alpha.
receptors on murine macrophages (C. Bogdan & A. Ding, J. Leukoc. Biol.,
52: 119 (1992); C. L. Manthey et al. (1992), supra). This might be
important in chronicsynovitis because prior work has demonstrated that
TNF-.alpha. plays a prominent role in CIA pannus proliferation (E. Brahn
et al., Lymphokine Cytokine Res., 11: 253 (1992)).
Adverse effects of Taxol appeared to be enhanced by the concurrent use of
AGM-1470, necessitating a reduction of Taxol dosing. However, as discussed
above, therapeutic. benefits may be maintained at lower dosage levels of
Taxol. Although few if any toxicities have been associated with long-term
administration of AGM-1470 (H. Brem & J. Folkman (1993), supra), Taxol
exposure can lead to multiple side effects, including neutropenia, major
hypersensitivity reactions, cardiac arrhythmias, and neurotoxicity (E. K.
Rowinsky et al., Sem. Oncol. 20 (Suppl. 3): 1 (1993)). Taxol treatment can
also cause mitotic arrest and cell necrosis in the gastrointestinal tract
(R. H. Hruban et al., Cancer 63: 1944 (1989)). An AGM-1470 interaction may
have enhanced Taxol's i.p. cytotoxic effect on cells undergoing mitosis,
resulting in the appearance of diarrhea and weight loss in the combination
protocol rats. These adverse effects were reversible over several days by
withholding treatment, probably aided by the rapid systemic clearance of
Taxol. Future dose scheduling changes could include a more aggressive
reduction of Taxol after the initial doses used in combination with
AGM-1470. This might achieve and maintain maximal early suppression of CIA
with minimal morbidity. Altering the sequence of drug administration may
also have an effect on the overall efficacy and toxicity of AGM-1470 and
Taxol co-administration. In a phase I study of Taxol and cisplatin, more
profound neutropenia and decreased Taxol clearance rates were noted when
Taxol administration followed cisplatin than with the alternate sequence.
(E. K. Rowinsky et al., J. Clin. Oncol. 9: 1692 (1991)). In a separate in
vitro study, optimum cytotoxic activity against L1210 leukemic cells was
demonstrated when Taxol preceded cisplatin than with the alternate
sequence or simultaneous drug administration (E. K. Rowinsky et al., J.
Cancer Res. Clin. Oncol. 119: 727 (1993)).
The inflammatory processes in human autoimmune diseases such as RA are
complex and involve both cellular and humeral immunity, inflammatory
mediators, and multiple cytokines (E. D. Harris, Jr., N. Eng. J. Med. 322:
1277 (1990)). The combination of two or more agents with different
targeted sites of action can provide more effective control of
inflammation. This is the first reported use of AGM-1470 and Taxol in
combination therapy. Significantly earlier and more effective suppression
of CIA inflammation was seen compared to either single agent alone. This
may be clinically important since early and aggressive control of
inflammation in RA patients may improve long term outcome as defined by
disability level and degree of irreversible joint damage (S. E. Gabriel &
H. S. Luthra, Mayo Clin. Proc. 63: 58 (1988); Y. S. Sherrer et al.,
Arthritis Rheum. 29: 494 (1986)) Further study of agents with distinct
and/or specific mechanisms of action in combination may lead to better
treatment options.
The results reported in this Example provides a novel scheme for combined
drug therapy for rheumatoid arthritis, and provides the basis for further
improvement in therapy.
ADVANTAGES OF THE INVENTION
The use of Taxol in the suppression of autoimmune rheumatoid disorders
provides another, significant, treatment for rheumatoid arthritis and
other autoimmune forms of arthritis. The use of Taxol can suppress or
ameliorate symptoms such as inflammation, swelling, abnormal
neovascularization, bone erosion, or cartilage erosion. Most
significantly, Taxol can be combined with other treatment methods, such as
the use of angiogenesis inhibitors. Taxol works rapidly to cause
regression or stabilization of symptoms, and is generally well tolerated.
The results reported suggest that the use of Taxol may be adaptable to
prophylaxis in susceptible individuals.
Although the present invention has been described in considerable detail
with regard to certain preferred versions thereof, other versions are
possible. Therefore, the spirit and scope of the appended claims should
not be limited to the descriptions of the preferred versions contained
herein.
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